Field of the Invention
The present invention relates to the formation of nanostructured plasmonic metal films on substrates, where such plasmonic films are useful for spectroscopy and immunoassays, and, in exemplary aspects, to surface-enhanced Raman scattering (SERS) and near-infrared fluorescence enhancing (NIR-FE) gold substrates that can be applied on substrates containing biological, organic, or other molecules to be assayed.
Introduction
The present invention relates to plasmonic gold substrates are used as microarray platforms for fluorescence enhanced, multiplexed immunoassay of proteins down to 0.01 pg/ml of 1 fM level over 6 logs of dynamic range. The proteins detected include antibodies, autoantibodies, protein biomarkers for diseases such as cancer, cytokines and other biological molecules. Protein microarrays on a nanostructured gold platform with NIR-FE enable rapid, high throughput immunoassays with sensitivities superior to ELISA and RIA. These platforms can be applied to a variety of biological molecules.
Identification of bimolecular interactions and further application of such interactions has and is making great contribution to both scientific research and clinical diagnostics, exemplified by Yalow and Berson's success in measuring insulin level in human serum which was realized through radio immunoassay (RIA) based on insulin-antibody interactions. Applications based on biomolecular interactions have flourished for several decades. For example, measurement of protein biomarkers such as carbohydrate antigen 125 (CA-125) and carcinoembryonic antigen (CEA) are clinically employed for therapeutic monitoring of ovarian cancer. Identification of human antibodies against autoantigens is helping doctors to diagnose/predict autoimmune disease such as rheumatoid arthritis (RA), system lupus erythematosus (SLE), etc. Measurement of human antibodies against certain antigens is also applied as a tool for monitoring human immunity against corresponding disease types, such as Influenza hemagglutination inhibition (HI) assay for evaluation of human immunity against flu.
The first generation of immunoassays for identification of biomolecules interactions was heavily reliant on radioactivity owe to its extraordinary sensitivity, while people are looking for alternatives to bypass the safety issues related to radioisotopes. Based on enzymatic reaction which changes the optical density of the substrate, enzyme-linked immunosorbent assay (ELISA) has become the gold standard for current immunoassays due to its high sensitivity and ease of use. However, accompanied by the quantum leap of genomic and proteomic project, large number screening of biomolecules interactions is becoming a necessity for scientists and clinicians nowadays, requiring a third generation of immunoassays with multiplex ability. Planar microarray assays and Luminex bead suspension assays are emerging as useful tools for high throughput biomolecules interactions screening. For planar substrate supported immunoassays, biomolecules are immobilized on planar substrate as probes and binding of biomolecules is reflected by the fluorescence intensity from the detecting reagent on corresponding probe “spots”. For Luminex bead assays, such probes are immobilized on polystyrene beads with unique fluorescence fingerprint and binding of biomolecules is also reflected by the fluorescence intensity from detecting reagent on corresponding bead. However, due to the same physical detection method, the sensitivity of traditional microarray and bead assays are no better than ELISA. Detecting biomarkers in serum resembles detecting needles in a haystack, as concentrations span up to nine orders of magnitude with relevant markers often present from nano-molar to femto-molar levels. Therefore assay sensitivity is an essential factor for evaluation of immunoassays besides multiplicity. Current planar microarray methodology is based largely on glass substrates or nitrocellulose substrates, with insufficient sensitivity for accurate protein marker quantification. Described below is a nanostructured gold (Au)-coated, plasmonic substrate capable of affording near-infrared fluorescence enhancement by ˜100-fold. Protein microarrays on such plasmonic Au substrates demonstrated highly sensitive detection of proteins such as carcinoembryonic antigen (CEA) down to ˜5 fM in whole serum, with a 6-order dynamic range. This plasmonic Au film is readily produced via a simple chemical method on a variety of substrates such as glass, affording fluorescence enhancement of NIR fluorophores by up to 100-fold. The ease-of-use and potential for rapid translation of this plasmonic protein chip technology may afford improvements in high-throughput screening of biomolecules interactions with great sensitivity.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Surface-enhanced Raman scattering (SERS) and near-infrared fluorescence enhancement (NIR-FE) effects provided by plasmonic substrates have been shown to vastly improve signal-to-noise ratios compared to traditional Raman scattering or fluorescence measurements, affording improvements to assays based upon the methodologies. Both enhanced spectroscopies are based on local field enhancement that occurs in the near vicinity of metallic nanoparticles when surface plasmon oscillations are driven for a specific optical wavelength. However, to date, preparation of highly stable plasmonic gold substrates requires complicated and expensive methodologies and instrumentation.
For example, utilizing the advantages of SERS, glucose, oligonucleotides, explosives and other analytes of interest have been detected at high sensitivity.[3-6] Recently, high sensitivity protein detection based upon bioconjugated single-walled carbon nanotube (SWNT) Raman labels and SERS in protein array format has been demonstrated.[7] However, preparation of the SERS-active substrate required undesirable vacuum deposition of gold films and thermal annealing of the assay substrates at 400° C.
Plasmonic SERS and NIR-FE-active substrates are often made by vacuum evaporation or sputtering,[7-9] high temperature annealing,[7] and Langmuir-Blodgett film transfer,[10] amongst other methods.[11-16] For many assays, especially those with biological components, it is desirable to produce plasmonic metal nanostructures without exposing the assay components to harsh conditions, such as high temperatures, organic solvents, and high vacuum. Deposition of desirable films from the aqueous phase circumvents many of the aforementioned problems, yet provides the opportunity to prepare large area, SERS and NIR-FE-active films.
Purely solution phase chemical synthesis of silver substrates has been reported for SERS and NIR-FE applications,[17] but Ag suffers from oxidation and instability problems, especially when reactive species are present, as is the case in bioassays. Gold films are promising as highly stable SERS substrates, and may be prepared from pre-made gold nanoparticle (Au NP) precursor seeds by reduction of chloroauric acid solution by hydroxylamine.[11, 18] However, deposition of pre-made Au NP seeds onto a substrate requires an amino- or mercaptosilane functionalized substrate, and thus the methodology is not directly amenable to polymeric or other complex surfaces, such as protein microarrays.[19]